We are developing live-attenuated, intranasal, pediatric vaccines against human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV). Building on years of molecular and biologic studies in the LID, we use reverse genetics systems of our design to produce highly defined vaccine candidates from cloned cDNAs. This provides well-defined vaccine viruses that can have improved properties and have attenuating mutations that can readily be monitored during manufacture and use. This also provides virus with a short, well-defined passage history that greatly reduces the chance of adventitious agents. The DNA intermediate also provides a stable vaccine seed and the means to modify the virus as necessary in response to clinical data. To date, the most promising live-attenuated RSV vaccine candidate, called rA2cp248/404/1030/delSH, exhibited genetic and phenotypic instability in clinical studies, and a more stable version would be desirable. Two missense attenuating mutations in the L protein were identified as being unstable, namely 831L and 1321N, with the latter mutation being the more unstable. We have therefore sought to stabilize these mutations in a version of rA2cp248/404/1030/delSH called MEDI-559. This involved evaluating the phenotypes of each of the possible 20 amino acids at each locus, and inspecting all possible attenuating codons to identify one requiring more than a single nucleotide change for de-attenuation. The need for 2 or 3 changes, rather than 1, would reduce the frequency of de-attenuation by orders of magnitude. We were unable to identify such a codon for the 831 locus, although we did identify a codon for which single-nucleotide reversion was somewhat less frequent. In contrast, for the 1321 locus, we did succeed in identifying an attenuating codon requiring 2 nucleotide changes to revert to a wt-like assignment. In vitro stress tests (passage at increasing temperature to force reversion) identified a second-site compensatory mutation at nearby position 1313. However, we were able to stabilize that position. These improved codons were inserted into MEDI-559 to create the new virus cps-2. In vitro stress tests confirmed that it had substantially increased stability, and a study in seronegative chimpanzees showed that the level of attenuation of cps2 was indistinguishable from that of MEDI-559. Thus, this provides an improved vaccine candidate. We investigated creating new attenuating mutations in RSV by deletion of one or more codons in the L protein. This was done with the expectation that deletion of a codon would be more refractory to reversion than a missense mutation. Evaluation of 17 different deletions at 5 sites failed to produce viable virus, suggesting that deletion of an amino acid from a protein is less well tolerated than a missense mutation. However, deletion of a sixth site, namely 1313, resulted in a virus that replicated efficiently at the permissive temperature of 32C, necessary for efficient vaccine manufacture, but was temperature-sensitive, attenuated, and stable during in vitro stress tests. This identified a new attenuating mutation with promising properties. This del1313 mutation is presently being combined with other stable mutations to make new vaccine candidates. For example, combination with deletion of the NS2 interferon antagonist gene provided a new candidate that, in seronegative chimpanzees, was comparable in attenuation to MEDI-559 and cps2 and thus is a desirable new candidate. RSV infects and causes severe disease more frequently and earlier in infancy compared to other common respiratory viruses, suggesting that it is less sensitive to restriction by maternally-derived serum antibodies. RSV also is able to re-infect symptomatically (but usually with reduced disease) throughout life without antigenic change, which also is suggestive of inefficient restriction by host immunity. The virus has two viral neutralization antigens, the G and F surface glycoproteins, and the former is expressed as both membrane-bound and secreted (sG) forms due to the use of two different translational start codons, ATG-1 and ATG-48. We previously showed that sG reduces antibody-mediated restriction in two ways: by acting as an antigen decoy, and by inhibiting the antibody-mediated antiviral effects of Fc receptor-bearing leucocytes. We hypothesized this latter effect might involve cell-types such as macrophages, neutrophils and natural killer cells that contribute to viral clearance by phagocytosis of antibody-antigen complexes or by antibody-dependent cell-mediated cytotoxicity (ADCC). Interference with complement-mediated antiviral effects also was possible. We therefore investigated antibody-mediated restriction of RSV and effects of sG. We found that sG did not directly inhibit phagocytosis by macrophages. It also did not detectably inhibit the activation of pulmonary macrophages in mice. We also found that natural antibodies (background antibodies produced without antigen stimulation) do not appear to play a role in restricting and resolving RSV replication in RSV-naive mice. Depletion experiments showed that neutrophils did not appear to be significantly antiviral, either in RSV-naive mice or in mice that had received passive RSV-neutralizing antibodies. Macrophage depletion experiments showed that this cell-type is important in restricting RSV replication, both in RSV-naive mice and in mice that were given passive RSV-neutralizing antibodies. Furthermore, sG interfered with antibody-mediated restriction by macrophages. Finally, depletion experiments showed that the complement system does not play a significant role in restricting virus in RSV-naive mice, but is important for antibody-mediated restriction and is subject to inhibition by sG. These results highlighted the importance of macrophages and complement in restricting RSV replication, and demonstrated the ability of sG to interfere with both mechanisms. Pneumonia virus of mice (PVM), a relative of RSV, causes respiratory disease in mice. There is serologic evidence suggesting widespread exposure of humans to PVM. To investigate replication in primates, African green monkeys (AGM) and rhesus macaques were inoculated with PVM by the respiratory route. Virus was shed intermittently at low levels by a subset of animals, suggesting poor permissiveness. PVM efficiently replicated in cultured human cells and inhibited the type I interferon (IFN) response in these cells. This suggests that poor replication in non-human primates was not due to a general non-permissiveness of primate cells or poor control of the IFN response. Seroprevalence in humans was examined by screening sera from 30 adults and 17 young children for PVM neutralizing activity. Sera from 6% of children and 40% of adults had low neutralizing activity against PVM, which could be consistent with increasing incidence of exposure following early childhood. There was no cross-reaction of human or AGM immune sera between RSV and PVM, and no cross-protection in the mouse model. In native Western blots, human sera reacted with RSV but not PVM proteins under conditions where AGM immune sera reacted strongly. Serum reactivity was further evaluated by flow cytometry using unfixed Vero cells infected with PVM or RSV expressing GFP as a measure of viral gene expression. The reactivity of human sera against RSV-infected cells correlated with GFP expression, whereas reactivity against PVM-infected cells was low and uncorrelated with GFP expression. Thus, PVM-specificity was not evident. Our results indicate that the PVM-neutralizing activity of human sera is not due to RSV- or PVM-specific antibodies but may be due to natural IgG antibodies. The absence of PVM-specific antibodies and restriction in non-human primates make PVM unlikely to be a human pathogen.